In human B cells infected with Epstein-Barr virus (EBV), latency-associated virus gene products inhibit expression of the pro-apoptotic Bcl-2-family member Bim and enhance cell survival. This involves the activities of the EBV nuclear proteins EBNA3A and EBNA3C and appears to be predominantly directed at regulating Bim mRNA synthesis, although post-transcriptional regulation of Bim has been reported. Here we show that protein and RNA stability make little or no contribution to the EBV-associated repression of Bim in latently infected B cells. However, treatment of cells with inhibitors of histone deacetylase (HDAC) and DNA methyltransferase (DNMT) enzymes indicated that epigenetic mechanisms are involved in the down-regulation of Bim. This was initially confirmed by chromatin immunoprecipitation analysis of histone acetylation levels on the Bim promoter. Consistent with this, methylation-specific PCR (MSP) and bisulphite sequencing of regions within the large CpG island located at the 5′ end of Bim revealed significant methylation of CpG dinucleotides in all EBV-positive, but not EBV-negative B cells examined. Genomic DNA samples exhibiting methylation of the Bim promoter included extracts from a series of explanted EBV-positive Burkitt's lymphoma (BL) biopsies. Subsequent analyses of the histone modification H3K27-Me3 (trimethylation of histone H3 lysine 27) and CpG methylation at loci throughout the Bim promoter suggest that in EBV-positive B cells repression of Bim is initially associated with this repressive epigenetic histone mark gradually followed by DNA methylation at CpG dinucleotides. We conclude that latent EBV initiates a chain of events that leads to epigenetic repression of the tumour suppressor gene Bim in infected B cells and their progeny. This reprogramming of B cells could have important implications for our understanding of EBV persistence and the pathogenesis of EBV-associated disease, in particular BL.

To explore the role of p16INK4a as an intrinsic barrier to B cell transformation by EBV, we transformed primary B cells from an individual homozygous for a deletion in the CDKN2A locus encoding p16INK4a and p14ARF. Using recombinant EBV-BAC viruses expressing conditional EBNA3C (3CHT), we developed a system that allows inactivation of EBNA3C in lymphoblastoid cell lines (LCLs) lacking active p16INK4a protein but expressing a functional 14ARF-fusion protein (p14/p16). The INK4a locus is epigenetically repressed by EBNA3C – in cooperation with EBNA3A – despite the absence of functional p16INK4a. Although inactivation of EBNA3C in LCLs from normal B cells leads to an increase in p16INK4a and growth arrest, EBNA3C inactivation in the p16INK4a-null LCLs has no impact on the rate of proliferation, establishing that the repression of INK4a is a major function of EBNA3C in EBV-driven LCL proliferation. This conditional LCL system allowed us to use microarray analysis to identify and confirm genes regulated specifically by EBNA3C, independently of proliferation changes modulated by the p16INK4a-Rb-E2F axis. Infections of normal primary B cells with recombinant EBV-BAC virus from which EBNA3C is deleted or with 3CHT EBV in the absence of activating ligand 4-hydroxytamoxifen, revealed that EBNA3C is necessary to overcome an EBV-driven increase in p16INK4a expression and concomitant block to proliferation 2–4 weeks post-infection. If cells are p16INK4a-null, functional EBNA3C is dispensable for the outgrowth of LCLs.

Detailed analyses of the chromatin around the BIM promoter has revealed that latent Epstein–Barr virus (EBV) triggers the recruitment of polycomb repressive complex 2 (PRC2) core subunits and the trimethylation of histone H3 lysine 27 (H3K27me3) at this locus. The recruitment is absolutely dependent on nuclear proteins EBNA3A and EBNA3C; what is more, epitope-tagged EBNA3C could be shown bound near the transcription start site (TSS). EBV induces no consistent changes in the steady-state expression of PRC2 components, but lentivirus delivery of shRNAs against PRC2 and PRC1 subunits disrupted EBV repression of BIM. The activation mark H3K4me3 is largely unaltered at this locus irrespective of H3K27me3 status, suggesting the establishment of a ‘bivalent’ chromatin domain. Consistent with the ‘poised’ nature of these domains, RNA polymerase II (Pol II) occupancy was not altered by EBV at the BIM TSS, but analysis of phospho-serine 5 on Pol II indicated that EBNA3A and EBNA3C together inhibit initiation of BIM transcripts. B cell lines carrying EBV encoding a conditional EBNA3C-oestrogen receptor-fusion revealed that this epigenetic repression of BIM was reversible, but took more than 3 weeks from when EBNA3C was inactivated.

Mature human B cells infected by Epstein-Barr virus (EBV) become activated, grow, and proliferate. If the cells are infected ex vivo, they are transformed into continuously proliferating lymphoblastoid cell lines (LCLs) that carry EBV DNA as extra-chromosomal episomes, express 9 latency-associated EBV proteins, and phenotypically resemble antigen-activated B-blasts. In vivo similar B-blasts can differentiate to become memory B cells (MBC), in which EBV persistence is established. Three related latency-associated viral proteins EBNA3A, EBNA3B, and EBNA3C are transcription factors that regulate a multitude of cellular genes. EBNA3B is not necessary to establish LCLs, but EBNA3A and EBNA3C are required to sustain proliferation, in part, by repressing the expression of tumour suppressor genes. Here we show, using EBV-recombinants in which both EBNA3A and EBNA3C can be conditionally inactivated or using virus completely lacking the EBNA3 gene locus, that—after a phase of rapid proliferation—infected primary B cells express elevated levels of factors associated with plasma cell (PC) differentiation. These include the cyclin-dependent kinase inhibitor (CDKI) p18INK4c, the master transcriptional regulator of PC differentiation B lymphocyte-induced maturation protein-1 (BLIMP-1), and the cell surface antigens CD38 and CD138/Syndecan-1. Chromatin immunoprecipitation sequencing (ChIP-seq) and chromatin immunoprecipitation quantitative PCR (ChIP-qPCR) indicate that in LCLs inhibition of CDKN2C (p18INK4c) and PRDM1 (BLIMP-1) transcription results from direct binding of EBNA3A and EBNA3C to regulatory elements at these loci, producing stable reprogramming. Consistent with the binding of EBNA3A and/or EBNA3C leading to irreversible epigenetic changes, cells become committed to a B-blast fate <12 days post-infection and are unable to de-repress p18INK4c or BLIMP-1—in either newly infected cells or conditional LCLs—by inactivating EBNA3A and EBNA3C. In vitro, about 20 days after infection with EBV lacking functional EBNA3A and EBNA3C, cells develop a PC-like phenotype. Together, these data suggest that EBNA3A and EBNA3C have evolved to prevent differentiation to PCs after infection by EBV, thus favouring long-term latency in MBC and asymptomatic persistence.

It is well established that Epstein-Barr virus nuclear antigen 3C (EBNA3C) can act as a potent repressor of gene expression, but little is known about the sequence of events occurring during the repression process. To explore further the role of EBNA3C in gene repression–particularly in relation to histone modifications and cell factors involved–the three host genes previously reported as most robustly repressed by EBNA3C were investigated. COBLL1, a gene of unknown function, is regulated by EBNA3C alone and the two co-regulated disintegrin/metalloproteases, ADAM28 and ADAMDEC1 have been described previously as targets of both EBNA3A and EBNA3C. For the first time, EBNA3C was here shown to be the main regulator of all three genes early after infection of primary B cells. Using various EBV-recombinants, repression over orders of magnitude was seen only when EBNA3C was expressed. Unexpectedly, full repression was not achieved until 30 days after infection. This was accurately reproduced in established LCLs carrying EBV-recombinants conditional for EBNA3C function, demonstrating the utility of the conditional system to replicate events early after infection. Using this system, detailed chromatin immunoprecipitation analysis revealed that the initial repression was associated with loss of activation-associated histone modifications (H3K9ac, H3K27ac and H3K4me3) and was independent of recruitment of polycomb proteins and deposition of the repressive H3K27me3 modification, which were only observed later in repression. Most remarkable, and in contrast to current models of RBPJ in repression, was the observation that this DNA-binding factor accumulated at the EBNA3C-binding sites only when EBNA3C was functional. Transient reporter assays indicated that repression of these genes was dependent on the interaction between EBNA3C and RBPJ. This was confirmed with a novel EBV-recombinant encoding a mutant of EBNA3C unable to bind RBPJ, by showing this virus was incapable of repressing COBLL1 or ADAM28/ADAMDEC1 in newly infected primary B cells.